Project Summary/Abstract Aging is a multifaceted process that encompasses changes in cellular quality control, antioxidant and anti- inflammatory mechanisms as well as in the overall cellular metabolism. The activity of mitochondria is tightly associated with the aging process. Decline in mitochondrial function results in a decrease in cellular oxidative capacity, an increase in the production of reactive oxygen species (ROS), and a corresponding increase in the mutation rate of mitochondrial DNA. The electron transport chain (ETC) plays an essential role in the observed metabolic effects of aging and comprises four multimeric complexes: complex I [CI, NADH:ubiquinone oxidoreductase], complex II [CII, succinate dehydrogenase], complex III [CIII, ubiquinol-cytochrome c oxidoreductase] and complex IV [CIV, cytochrome c oxidase]. With the exception of CII that is fully nuclear encoded, the ETC complexes (CI, CIII and CIV) contain both nuclear and mitochondrial encoded subunits. Of the 13 proteins encoded by mitochondrial DNA (mtDNA), 11 are components of the respiratory chain while the other two are membrane subunits of the F-ATPase. The interaction between the ETC complexes as they form higher order assemblies, known as supercomplexes, are hypothesized to minimize the production of ROS by optimizing the flow of substrates between enzymes. Previous studies have shown a moderate decrease in the activity of the individual complexes and amount of supercomplexes with age, but this does not fully account for the observed mitochondrial dysfunction. We hypothesize that the specific conformation of the supercomplexes changes with age and thereby the efficiency of the interaction between complexes decreases. Some of the potential structural changes, including those of lipid molecules tightly bound to CI and CIII, are not easily detectable using current biochemical methods. We propose to investigate the 3D structures of the CI1CIII2CIV supercomplex using electron microscopy to elucidate the role of aging on the strength of interactions between these complexes. In this process, methods for the purification of supercomplexes from heart muscle of both young and old mice will be optimized and the activities and ratios of single enzymes and enzymes integrated into supercomplexes will be quantified. Additionally, we propose to investigate the 3D structures of transient mitochondrial respiratory chain supercomplexes. Determination of the structural differences will aid in understanding the decline of energy metabolism with aging. Completion of the proposed aims will provide a solid foundation for future studies to dissect how modest architectural differences in supramolecular organization can lead to extreme functional defects and will reveal novel means to combat senescence.